Chromosomal targeting in bacteria using FLP recombinase

ABSTRACT

A method of introducing exogenous cloned DNA into a bacterial chromosome of a bacteria in which the transposon Tn5 and the FLP recombinase are functional in vivo is disclosed. In one embodiment, the method comprises the steps of: (a) introducing FLP recombination target sites (FRTs) permanently at random locations in a bacterial chromosome using a plasmid vector that contains an FRT within a modified Tn5 transposon, two selectable markers, and a removable replication origin; (b) mapping the FRT introduced into the bacterial chromosome; (c) cloning exogenous DNA into a vector comprising two FRT sites, two selectable markers, and a removable replication origin; (d) removing the replication origin in the vector of step (c); (e) introducing the altered plasmid vector into bacterial cells, wherein the bacteria cells comprise a functional FLP recombinase; and (f) obtaining targeted integrants.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies: NIH Grant Nos.: GM37835; AI00599; GM32335;GM52725; and NSF Grant No.: MCB-9600715.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Ser. No. 60/059,128,filed Sep. 17, 1997.

BACKGROUND OF THE INVENTION

Site-specific recombination provides a vehicle to introduce exogenousDNA, delete DNA, or rearrange DNA at specific sites in a chromosome(41). Among the site-specific recombination systems characterized todate, the FLP system of the yeast 2 micron plasmid and the Cre-loxsystem of bacteriophage P1 are among the most attractive for genomicmanipulation because of their efficiency, simplicity, and demonstratedin vivo activity in a wide range of organisms. These systems have beenused to construct specific genomic deletions and gene duplications,study gene function, promote chromosomal translocations, promotesite-specific chromosome cleavage, and facilitate the construction ofgenomic libraries in organisms including bacteria, yeast, insects,plants, mice, and humans 2-5, 10-18, 24-26, 28, 30-35, 38-41, 44, 45,47, 50). These studies have only begun to tap the potential of theapproach.

Site-specific recombination catalyzed by the FLP and Cre recombinasesoccurs readily in bacterial cells (1, 5, 6, 21, 33). In principle, itcould find wide application to studies of genomic structure and functionas well as enhance the usefulness of E. coli in biotechnology.Ironically, this approach has not been exploited in bacteria as it hasbeen in eukaryotes, although bacteria were the first non-yeast cells inwhich FLP-mediated recombination was demonstrated (6). Even though genetargeting in bacteria can be achieved by homologous recombination,chromosomal targeting by site-specific recombination provides a newroute to stable transformation with the advantages of very highefficiency, defined reproducible insertion sites in the chromosome, andcontrolled reversibility.

The yeast FLP system has been studied intensively (7, 8, 22, 36). Theonly requirements for FLP recombination are the FLP protein and the FLPRecombination Target (FRT) sites on the DNA substrates. The minimalfunctional FRT site contains only 34 bp. The FLP protein can promoteboth inter- and intra-molecular recombination.

Previously, the inventors (Huang, et al., 1991) reported theconstruction of a model system in E. coli using the FLP recombinationsystem for chromosomal targeting and demonstrated the effectiveness ofthe general approach (21). The site-specific integration was absolutelydependent upon the expression of FLP protein and the presence of FRTsites in the chromosome. In some experiments, from 1% to 10% of theexogenous DNA molecules used, introduced on a modified bacteriophage λvector, actually found their way into a cell and were integrated intothe chromosome specifically at a chromosomal FRT.

Although Huang, et al. (1991) achieved a high integration frequency inthis original targeting system, there were limitations inherent to theconstructs that precluded a detailed characterization as well as aconvenient application of the system to bacterial cloning and genomicstudies.

BRIEF SUMMARY OF THE INVENTION

We have modified previous FLP systems to provide a method that canregulate and monitor excision as well as integration, introduce FRTtargets virtually anywhere in the chromosome and test a variety ofadditional parameters that might affect integration and/or excision.This is designated the FLIRT system, for "FLP-mediated DNA integrationand rearrangement at prearranged genomic targets."

In one embodiment, the invention is a method of introducing exogenouscloned DNA into a bacterial chromosome of a bacteria in which thetransposon Tn5 and the FLP recombinase are functional in vivo. Themethod comprises the steps of: (a) introducing at least one FLPrecombination target site (FRT) permanently at random locations in abacterial chromosome using a plasmid vector that contains an FRT withina modified Tn5 transposon, two selectable markers, and a removablereplication origin; (b) mapping the introduced FRT; (c) cloningexogenous DNA into a vector comprising two FRT sites, two selectablemarkers, and a removable replication origin; (d) removing thereplication origin in the vector of step (c); (e) introducing thealtered plasmid vector into bacterial cells, wherein the bacteria cellscomprise a functional FLP recombinase; and (f) obtaining targetedintegrants.

In a preferred version, the bacteria is a gram negative bacteria, mostpreferably an E. coli.

In another preferred embodiment, FLP recombinase is used in step (d) toremove the origin of replication.

In another preferred embodiment, more than one FRT is introduced intothe bacterial chromosome.

The present invention is also a system of plasmid constructs designed toallow very convenient use of the overall targeting scheme. Onepreferable plasmid comprises: (a) at least one FRT site; (b) aselectable marker located between two outside ends of transposon Tn5;and (c) a removable replication origin.

In another embodiment, the invention is a plasmid useful in cloningexogenous DNA in the method of described above. Preferably, the plasmidcomprises (a) at least two FRT sites; (b) two selectable markers; (c) aremovable replication origin; and (d) at least one restriction sitepositioned for use as a cloning site.

Most preferably, the plasmid of the present invention is selected fromthe group consisting of plasmid pEAW127, plasmid pEAW133, plasmidpEAW116, and plasmid pEAW1135.

It is an advantage of the present invention that a FLP cloning system isdescribed that provides means to regulate and monitor excision andintegration of a heterologous gene in a bacterial chromosome.

Other advantages, features and objects of the present invention willbecome apparent to one of skill in the art after review of thespecification, drawings and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A, B, and C is a diagram of the FLIRT system. FIG. 1A describesplasmids used to introduce FRT sites into the chromosome. FIG. 1Bdiagrams FLP expression plasmid pLH29. FIG. 1C diagrams plasmids used tointroduce exogenous DNA into bacteria and target it to chromosomal FRTsites.

FIG. 2 is a scheme for introducing FRT sites into the bacterialchromosome.

FIG. 3 diagrams the mapped locations of some FRT targets generated bypEAW127.

FIG. 4 is a scheme for targeting exogenous DNA to the chromosomal FRTs,using pEAW116 and derivatives.

FIGS. 5A and B is a demonstration of FLP-mediated site-specificchromosomal targeting with the FLIRT system and shows Southern analysesof successive steps in targeting. FIG. 5A is directed to introduction ofFRT target #3. FIG. 5B is directed to introduction of FRT target #4.FIG. 5C shows the sensitivity to UV irradiation of the strains analyzedin FIG. 5B, lanes 1-4.

FIG. 6 graphs rates of FLP-mediated excision of targeted DNA from achromosomal FRT site.

FIG. 7 is a diagram of pEAW135.

BRIEF DESCRIPTION OF THE INVENTION

In General

We have created a system that utilizes the FLP recombinase of yeast tointroduce exogenous cloned DNA reversibly at defined locations in the E.coli chromosome. (This system is described in Huang, et al. (1997),hereby incorporated by reference.) Recombination target sites (FRTs) canbe introduced permanently at random locations in the chromosome on amodified Tn5 transposon, now designed so that the inserted FRT can bedetected and its location mapped with base pair resolution. FLPrecombinase is provided as needed through the regulated expression ofits gene on a plasmid. Exogenous DNA is introduced on a cloning vectorthat contains an FRT, selectable markers, and a replication origindesigned to be deleted prior to electroporation for targeting purposes.High yields of targeted integrants are obtained, even in a recAbackground.

The system of the present invention permits a rapid and precise excisionof the introduced DNA when needed, without destroying the cells. Theefficiency of targeting appears to be affected only modestly bytranscription initiation upstream of the chromosomal FRT site. With rareexceptions, FRTs introduced to the bacterial chromosome are targetedwith high efficiency regardless of their location. The system shouldfacilitate studies of bacterial genome structure and function, simplifya wide range of chromosomal cloning applications, and generally enhancethe utility of E. coli as an experimental organism in biotechnology.

In one embodiment, the present invention is a method of introducing aforeign gene at defined locations in a bacterial chromosome. The methodcomprises the steps of obtaining a bacterial population, preferably anE. coli population, in which recombination target sites (FRTs) have beenintroduced permanently at random locations in the chromosome, preferablyon a modified Tn5 transposon. In a most preferred form of the presentinvention, the inserted FRT has been detected and the location mapped.

The natural full-length Tn5 is 5818 bp in length. The plasmid wedescribe comprises a "modified" Tn5, which is about 1200 bp altogether.The parts necessary for a suitable vector are (1) 112 bp of the outsideends of the transposon (this is the only part of Tn5 within the plasmidsection that is transferred to the bacterial chromosome), whichcomprises the two 56 bp segments that contain the sites that are boundby the transposase enzyme; and (2) the gene encoding the Tn5 transposaseenzyme (about 1100 bp altogether). This gene is expressed and promotesthe movement of the segment we are interested in from the plasmid to thechromosome. The 56 bp segments begin at the end of the transposon andextend inward for 56 bp. These segments are described in Huang, et al.(1997) and Reznikoff (1993). The plasmid itself does not replicate andhence exists only transiently in the cell. Because the transposase geneis not transferred, the transferred segment cannot come out of thechromosome once the plasmid disappears.

If one wished to use another transposon, one would need to takecorresponding elements from the candidate transposon to create a vectoruseful for the present invention.

To be introduced into the chromosome, the FRT must be between theoutside ends of Tn5 (between 40 and 50 bp, preferably 46 bp of DNA, foreach outside end). The transposase will catalyze the movement of anyelement between these ends. The transposase enzyme is supplied byexpressing its gene, also located on the plasmid but not within theoutside ends. The plasmid is brought into the cell by standard methodsof bacterials transformation (Typically, DNA and prepared cells aresimply mixed under conditions where a few of the cells spontaneouslytake up the plasmid). Once in the cell, the transposase gene isexpressed, transposase enzyme is synthesized, and the transposase enzymethen promotes the movement of the DNA segment between the outside endsfrom the plasmid into the chromosome.

A foreign gene is then introduced on a cloning vector that contains anFRT and a selectable marker. The pEAW127 and 133 constructs (describedbelow) used to put the FRT on the chromosome are designed to make iteasy to determine the exact location of the FRT on the chromosome. Thecloning vectors used to target the chromosomal FRT are the pEAW116 andpEAW135 constructs (also described below). These cloning vectors containa replication origin designed to be deleted prior to electroporation fortargeting purposes.

For pEAW116 and pEAW135, which bring in exogenous DNA and target it tothe chromosomal FRT, one only needs some cells already containing achromosomal FRT, the vectors (with the desired DNA already inserted intothem), and an enzyme to remove the replication origin on the vectors.Two steps in the overall process can be promoted by the FLP recombinase.The segment of the plasmid containing the replication origin is removed.(With pEAW116, this is done with purified FLP recombinase; with pEAW135,it can be done with the restriction enzyme XbaI plus DNA ligase). Theabbreviated plasmid DNA is then introduced in the cells by thetransformation method described above. Once inside the cell, FLPrecombinase is used again to introduce the entire abbreviated plasmidinto the chromosome at the site of the FRT. The FLP recombinase used inthis latter step is not purified, but instead is expressed from a geneencoding the FLP recombinase present on a separate plasmid (pLH29).

An enzyme capable of removing the replication origin, preferably the FLPrecombinase, is provided, preferably in a purified form. Once thecloning vector is introduced into the bacterial cell, an enzyme capableof integrating it into the chromosomal FRT site, preferably the FLPrecombinase, is provided, preferably as a plasmid construct. Thetargeted integrant is then obtained.

The FLP recombinase gene is found on the yeast 2 micron plasmid, aplasmid that is widely distributed in natural strains of the yeastSaccharomyces cerevisiae. Cox (1983) describes standard cloning methods.

In a most preferable form of the present invention, the plasmidsdescribed below are used to insert the FRT site in the bacterialchromosome, introduce exogenous DNA, and provide FLP recombinase.

The two plasmids in the FLIRT system are likely to be useful in almostall gram-negative bacteria, a class that includes a range of importanthuman pathogens. The only component described below that does not have awide host range is the plasmid pLH29, used to express the FLPrecombinase. If the FLP recombinase were expressed in a broad host rangeplasmid, such as a vector using the RK2 replicon, the entire systemcould be used in most, if not all, gram negative bacteria.

EXAMPLES

1. Materials and Methods

Media. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was fromIndofine. IPTG (isopropyl-β-D-thiogal-actopyranoside) was purchased fromBachem. Antibiotics were purchased from Sigma. Bacterial strains weregrown at 37° C. or 30° C. in L broth or on agar plates prepared asdescribed (29) and supplemented with antibiotics as appropriate.Antibiotic concentrations were: ampicillin (Amp), 100 mg/l for cellscontaining multiple copies of the β-lactamase gene on plasmids or 20mg/l for cells containing a single copy of the β-lactamase gene on thechromosome; kanamycin (Kan), 40 mg/l; tetracycline (Tet), 15 mg/l;chloramphenicol (Cam), 25 mg/l; X-Gal, 40 mg/l. IPTG was used at 0.5 mMor 1 mM as indicated.

Bacterial strains. Key parental strains employed in this work are: CSH26[F⁻ ara Δ(lac pro) thi] (29); RZ211 [F⁻ ara Δ(lac pro) thi srl recA56](23); and MG1655 (F⁻, wild-type) (19). The strains RZ211 and MG1655 wereobtained from W. Reznikoff (University of Wisconsin) and G. Weinstock(University of Texas-Houston), respectively.

Enzymes and Reagents. Restriction enzymes and bacteriophage T4 DNAligase were obtained from New England Biolabs, Promega, or BoehringerMannheim. The FLP recombinase was purified and stored as describedelsewhere (46). AMV reverse transcriptase was from Life Sciences. VentDNA polymerase, Klenow fragment and linkers were purchased from NewEngland Biolabs. Sequencing of DNA in experiments involving the FLIRTsystem was performed using the Sequenase version 2.0 DNA sequencing kitfrom Amersham Life Science. Bacterial alkaline phosphatase, BIONICKlabeling system, and PHOTOGENE Nucleic Acid detection system were fromGibco BRL. Linkers and biotinylated lambda HindIII digest were from NewEngland Biolabs. All enzymatic reactions were performed essentially asdescribed by Sambrook, et al. (37) or as recommended by the suppliers.Radiolabeled deoxynucleotide triphosphates were obtained from AmershamLife Science. Oligonucleotides were prepared at the DNA synthesisfacility in the University of Wisconsin Biotechnology Center or werefrom Operon Technologies Inc. SeaPlaque agarose was from FMCBioProducts. GENECLEAN was from Bio 101 Inc. Transformation and Bochnerselection media were as previously described (9). All chemicals were ofanalytical grade or better and were purchased from common vendors.

FLIRT System Vectors.

(i) Vectors for the delivery of exogenous DNA. A cloning vector forconvenient targeting of exogenous DNA to the bacterial chromosome ispEAW116. To create pEAW116, pJFS36 (42) was digested with BamHI, theends were filled in, and ClaI linkers were ligated at this site. Theresulting construct was digested with ClaI and SphI. A synthetic DNAfragment, with ClaI and SphI ends, containing a short polylinker withHindIII, PstI and SalI sites and an FRT in the same orientation as theFRT already present in pJFS36, was ligated to the prepared vector. Theresulting plasmid (now with two SalI sites) was partially digested withSalI and the ends filled in. The tetracycline resistance gene from Tn10was ligated to the filled-in SalI site, and a construct was chosen inwhich the Tet^(R) gene had been inserted in the polylinker between theFRTs. This plasmid was then isolated and digested at HindIII. A shortsynthetic polylinker containing one HindIII sticky end, sites for KpnI,SmaI, NotI, NheI, BglII, and another end compatible with HindIII butthat does not regenerate a HindIII site after ligation (so that HindIIIappears at one but not both ends of the fragment added to the vector),was ligated into the cleaved vector. This plasmid is pEAW116. A variantof pEAW116 was constructed by digesting it at the polylinker SmaI andNheI sites and filling in the ends, and then inserting the wild-typerecλ gene and its promoter to generate pEAW118.

(ii) Plasmids for introducing FRT sites on the E. coli chromosome.Plasmid pEAW127 contains an FRT, selectable marker, and polylinkerbetween 56 bp Tn5 outside end (OE) sequences, 19 of which are requiredfor Tn5 transposition. The ampicillin resistance gene, origin ofreplication, lacIq, and Tn5 transposase are located in the region thatis not transposed. The FRT was the minimal wild-type FRT derived frompJFS36 (42). The starting point was plasmid pRZ4828 (Reznikoffunpublished work), constructed by inserting a mini-Tn5 element,containing 56 bp of the Tn5 outer ends and BamHI sites flanking theTn903 kanamycin resistance gene, into the filled-in BamHI site ofpRZ4825 (48). The Tn903 Kan^(R) gene of pRZ4828 was first deleted bydigestion with BamHI. This was replaced with a BamHI fragment with bases1052-2262 of Tn903 containing the kanamycin resistance gene, a shortpolylinker containing NotI, KpnI, and DraIII sites, and an FRT, ligatedto the BamHI digested pRZ4828. This places all of the sequences betweenthe Tn5 outer ends. In order to facilitate the deletion of thereplication origin prior to electroporation, EcoRI sites were placed oneither side of the plasmid origin (EcoRI linkers were placed at thefilled-in NdeI site, and at the DraI site that is not included in theAmp^(R) gene).

(iii) Plasmids for FLP expression. The plasmid pLH29 provides forexpression of FLP recombinase, regulated by plac along with an integrallacI gene. Construction of this plasmid is described elsewhere (20).

Construction of target strains. MG1655 srl::Tn10 Δrecλ1398 wastransformed with pLH29 and selected for chloramphenicol resistance.Tet^(S) mutants were then selected using Bochner medium (9). These weredesignated MG1655 ΔrecA Tet^(S) pLH29. The plasmid pEAW127 (10 μg) wasdigested with EcoRI to remove the origin of replication. In order toseparate any contaminating undigested pEAW127, the digested DNA wasincubated at 65° C. for 10 minutes with an equal volume of 1.5%SeaPlaque low-melting point agarose. This was then loaded in the wellsof a horizontal 20 cm long, 0.8% agarose gel and allowed to cool 5minutes before the 1×TAE buffer was added and the gel was run. Thelow-melting point agarose matrix trapped the circular DNA in the wells(FMC BioProducts, Hank Daum, III personal communication). The largeEcoRI fragment without the origin was excised from the gel, and DNA waseluted using Geneclean. The DNA was self-ligated to circularize for 1hour at room temperature in a volume of 65 μl. The ligation mix wasextracted once with an equal volume of phenol: chloroform: isoamylalcohol (25:24:1) and ethanol precipitated. The resulting circular DNAwas resuspended and digested with BspLU11I for 1 hour at 37° C. in avolume of 100 μl. This linearizes any contaminating pEAW127 that onlycut once with EcoRI, since the BspLU11I site is between the EcoRI sites.The BspLU11I digest was extracted once with phenol: chloroform: isoamylalcohol (25:24:1) and ethanol precipitated.

The resulting pEAW127Δori DNA was resuspended in 30 μl H₂ O. The DNAconcentration was determined from the OD260 and 0.2, 0.4, and 0.6 μg DNAwere electroporated into 40 μl of electrocompetent MG1655 Δrexλ Tet^(S)/pLH29 cells. Electrocompetent cells were grown in 0.5 mM IPTG, 25 mgCam/l and prepared according to the procedure from Bio-Rad.Electroporations were performed at 25 μf, 2.5 kV, and 200 Ohms in anice-cold cuvette, with a 0.2 cm gap, by a Bio-Rad Gene Pulser. The cellswere plated on 40 mg/l Kan plates and incubated at 37° C. overnight.Twenty-four Kan^(R) colonies were picked and screened on Amp plates.Kan^(R) Amp^(S) colonies indicate that a transposition event occurred toinsert the FRT and Kan^(R) gene onto the chromosome. Small-scale plasmidDNA preparations were done to confirm the presence of pLH29 as the onlyplasmid in the cells.

Target strains containing FRT sites located at pre-defined sites in thelac operon were generated by homologous recombination. These strainswere used to study the effect of transcription on targeting efficiency,with transcription regulated by IPTG. In these experiments, FLPexpression was provided by the plasmid pEAW38, in which the FLP gene issubject to temperature induction (21). For better control of the timingof the IPTG-mediated transcription, a lacy strain was preferred in thestudy. To obtain lacZ: :FRT lacY construct, strains RR1 (lacZ+lacY⁻) wasfirst transduced to recD::Tn10 by bacteriophage P1 grown on MG1655recD::Tn10 (from P. Kiley, University of Wisconsin). Then strainsRR1recD and MG1655recD were transformed with ScaI-linearized pLH20 andpLH32, respectively, by electroporation using a Bio-rad Gene Pulser andthe protocol recommended by the manufacturer. On the plasmids pLH20 andpLH32, the FRT sites were cloned within the lac operon, so the lac-FRTconstructs could replace the original lac sequence on the E. colichromosome by homologous recombination. The construction of theseplasmids is described elsewhere (20). In the case of pLH20transformation, LacZ⁻ colonies were screened on IPTG-XGal-LB plates; inthe case of pLH32 transformation, LacZ+LacY⁻ colonies were screened onlac-MacConkey plates. The colonies with desired phenotypes were picked,and the chromosomal FRT sites were transduced to wild-type RR1 (forpLH20) and MG1655 (pEAW38) (for pLH32). The colonies were selected forKan^(R) and screened for the Lac phenotype. The pLH20 FRT site in RR1was further transduced to wild-type MG1655 (pEAW38). The final strainderived from pLH20 transformants with the FRT site at lacZ is calledMG1655lacZ::FRT(pEAW38); and the final strain derived from pLH32transformants with the FRT site at lacY is calledMG1655lacY::FRT(pEAW38). Phenotypically MG1655lacZ::FRT is LacZ-LacY⁻,and MG1655lacY::FRT is LacZ+LacY⁻. The lac locations of the FRT siteswere confirmed by Southern analysis (data not shown).

Mapping the genomic location of FRT sites in target strains. Genomic DNAfrom the target strains was isolated as described (51), and 5 μg wasdigested with either NheI, PvuII, or SphI for 2 hours at 37° C. Thereare no sites for these enzymes in the FRT-containing DNA transposed tothe chromosome. The digested genomic DNA was extracted once with phenol:chloroform: isoamyl alcohol (25:24:1) and ethanol precipitated. Thegenomic DNA was ligated to pUC119 digested with XbaI, SmaI, or SphI,which generate ends compatible with NheI, PvuII, and SphI respectively.The ligated DNA was transformed into competent DH5a cells [supE44ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1], platedon 40 mg/l Kan plates, and incubated at 37° C. overnight. Kan^(R)colonies should contain inserts of transposed DNA and flanking genomicDNA. Plasmid DNA from the selected colonies was purified and sequencedusing a primer consisting of bases 1091-1074 of Tn903. This readsthrough the FRT and 56 bp Tn5 outer ends and into the flanking genomicDNA. The Fasta program (GCG) was used to compare the genomic DNAsequences with those in Genbank, and identify the precise locations ofthe FRT-containing sequences in seven target strains. These weredesignated MG1655 ΔrecA Tet^(S) 127FRT#1-#7/pLH29.

The insert for #1 maps next to base 67128 of the lambda clone accession#U29579 comprising the E. coli chromosomal region from 61 to 62 minutes.This is between two unidentified ORFs, o191 and f297. #2 transposed nextto base 228222 of the lambda clone with accession number U14003, at 92.8to 00.1 minutes. This is in unidentified ORF f326b. #3 transposed nextto base 2165 of the nagC gene which is at 15.5 minutes on the E. colichromosome. #4 transposed next to base 49628 of the lambda clone withaccession number U00039 comprising the E. coli chromosomal region from76-81.5 minutes. It is in unidentified ORF o383. #5 transposed next tobase 11640 of the clone with accession number D90699 at 12.6-12.9minutes on the chromosome. It is in the unidentified ORF o110. #6 mapsnext to base 51888 of the lambda clone with accession number U18997containing the region from 67.4 to 76.0 minutes. It is in theunidentified ORF f408. #7 maps next to base 19887 in accession numberU28379, at approximately 68 minutes. It is in the unidentified ORF f168.

Targeting Trials. The plasmids pEAW116 or pEAW118 were first linearizedby FspI digestion. Contaminating undigested plasmid DNA was separated bytrapping the circular DNA in 1.5% SeaPlaque low-melting point agarose asdescribed above for pEAW127. The linearized DNA (16.8 μg) was thenre-circularized by intramolecular FLP-mediated site-specificrecombination. The reaction mixture contained 25 mM N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer (pH 8),1 mM ethylenediamine tetraacetic acid (EDTA), 2.5 mg/ml bovine serumalbumin (BSA), 7.5% PEG 8000, 10.8% glycerol, 180 mM NaCl, and 145 nMFLP recombinase in a total reaction volume of 400 μl, and was carriedout at 30° C. for 10 minutes. The reaction was stopped by the additionof 50 μl of a solution containing 30% glycerol, 0.03% bromphenol blue,30 mM EDTA, 4% SDS. The reaction was loaded on a 0.8% agarose gel at 20μl/lane and run in 1×TAE (27). The circular deletion product of the FLPreaction was eluted from the agarose (Geneclean) in 20 μl H₂ O. The DNAconcentration was determined by absorption at 260 nm. This DNA (0.03-0.1μ was electroporated into electrocompetent MG1655 ΔrecA Tet^(S) 127FRT#3or #4/pLH29 cells as described above for processed pEAW127.Electroporated cells were selected for Tet^(R), then picked and screenedfor Tet^(R) and Amp^(R). Tet^(R), Amp^(S) colonies indicate targeting.To demonstrate the dependence of targeting on the presence of the FLPprotein and a chromosomal FRT, the same procedure was used toelectroporate electrocompetent cells of MG1655 ΔrecA Tet^(S)127FRT#4/pLH29 or Mg1655 ΔrecA Tet^(S) /pLH29, and MG1655 ΔrecA Tet^(S)127FRT#4 with processed pEAW118.

Reversibility of Targeting. In a typical experiment, single coloniesresulting from targeted integration were isolated. Overnight cultures ofthese isolates were diluted 100-fold in L broth containing 1 mM IPTG toinduce excision or no IPTG as a control. The cells were kept at 30° C.Once the culture reached stationary state (about 6 generations), analiquot of the culture was transferred to fresh media with 1:100dilution to resume the growth. At the same time, 0.1 ml of the culturewas plated on Amp selective media to determine the number of the cellswhich still kept the phenotype of an integrant. Also, the same volume ofthe cultures was plated on LB plates to determine the total number ofthe cells. The ratio of Amp^(R) surviving integrants vs. total cellswere determined. The excision rate (X) was calculated from the equation:X=1-e.sup.(ln(r)/n), where r=no. of Amp^(R) colonies/no. of totalcolonies and n=number of generations.

Testing for the presence of a functional recA gene by exposure to U.V.light. Overnight-cultured cells to be tested were diluted 1/100 in LBmedia, and grown for ˜1.5 hours to mid-log phase. Cultures (10 ml) werethen spun down at 2000 g, washed, resuspended in M9 media atOD600=0.054. Aliquots (2 ml) were placed on sterile uncovered 35 mmplates and shaken gently beneath a UV light source (254 nm). Theirradiation was conducted under a photographic red light to preventphoto-reactivation. Irradiation was carried out for an appropriate timeat fluence rate 0.8 J/m² s or 1.6 J/m² s. The lamp was calibrated beforeeach experiment using a J-225 Short Wave UV meter. The exposed (orunexposed control) cells were serially diluted and spread on TYE-Cam20plates. The plates were then wrapped in tinfoil and incubated overnightat 37° C. Colonies were counted the following day. Each data pointrepresents an average from two experiments.

Southern analysis. Genomic DNA (5 μg) was digested with 20 units ofPvuII for 2 hours at 37° C. in a final volume of 50 μl. The DNA wasethanol precipitated and resuspended in 10 μl TE. Each digested DNA wasloaded on a 1% agarose gel, along with 1 μl of a 100 μg/ml biotinylatedHindIII-digested lambda DNA marker. The gel was run at 50 milliamps in1×TAE, then photographed after staining in ethidium bromide. The DNA wastransferred to PhotoGene nylon membranes and Southern analysis wasperformed using the procedure specified by Gibco BRL. Probes were madeby excising the FRT, kanamycin resistance, and flanking genomic DNA fromthe subclones in pUC119 used for mapping the FRTs on the MG1655 ΔrecλTet^(S) chromosome. EcoRI and HindIII were used for the digestion, andthe DNA fragments were separated on a 0.8% agarose gel. The DNA to beused as a probe was eluted from the gel using GENECLEAN, and 1 μg DNAwas labeled using the Gibco BRL BIONICK labeling system.

2. Results

We previously described a method for chromosomal targeting of exogenousDNA in E. coli (21). The present work was undertaken to determine theeffects of parameters such as transcription, chromosomal location, andthe host homologous recombination system on integration efficiency, andto refine the system both to facilitate these experiments and to makethe system convenient for general use. The system consists of threeelements (I) E. coli target strains, each with a single FRT site locatedon the chromosome; (II) a plasmid permitting the regulated expression ofFLP protein; and (III) a delivery vector for exogenous DNA, containing adrug resistance gene marker as well as an FRT site compatible with theFRT site on the chromosome. We first describe the FLIRT system, which isentirely plasmid-based and designed for general use. We then brieflysummarize some results obtain ed with a variant of the bacteriophageλ-based system we reported on previously (21), which investigate some ofthe parameters that might affect targeting (20).

(i) The FLIRT system. The FLIRT system is presented in FIG. 1. FIGS. 1A,B, and C is a diagram of the FLIRT system. A) Plasmids used to introduceFRT sites into the chromosome. Important feature s are described in thetext. The plasmid pEAW133 differs from pEAW27 only in the addition of asecond FRT site flanking the Kan^(R) marker. After introduction of thetransposed segment to the chromosome and mapping, the additional FRTpermits the easy deletion of the Kan^(R) marker so that Kan selectioncan be used for other purposes. B) FLP expression plasmid pLH29. Unlikethe other components of the system, the plasmid pLH29 contains sequencesleft over from earlier constructs that are not necessary for (but do notinterfere with) its function. C) Plasmids used to introduce exogenousDNA into bacteria and target it to chromosomal FRT sites. The plasmidpEAW116 is designed as a general cloning vector, with a polylinkercontaining a number of unique restriction sites. The plasmid pEAW135,described in the text, is essentially the same as pEAW116, but lacksXbaI sites other than those within the FRT sites. With pEAW135, the partof the plasmid containing Tet^(R) and any cloned DNA can be removed andcircularized with XbaI plus ligation, eliminating any requirement forpurified FLP recombinase. For regulated FLP protein expression, theFLIRT system uses pLH29, which was described in earlier work (20, 21).The improvements in the FLIRT system involve the methods used tointroduce FRT sites into the E. coli genome, and to target exogenous DNAto those sites. The system also makes economical use of the most commonselectable markers.

(ii) Generating E. coli strains with FRT sites in the genome. Theplasmid pEAW127 includes one FRT site and a selectable marker (Kan^(R))located between the two outside ends (OE) of transposon Tn5 (FRTsegment). The Tn5 transposase is encoded elsewhere on the plasmid, andis not transferred to the bacterial chromosome with the FRT site. Thesite is thus stable and permanent once it is transferred. Thereplication origin is removed from the plasmid prior to electroporation.Since the plasmid DNA is introduced into the cell only transiently,stable transformation to Kan^(R) requires the transposition of the FRTsegment to the bacterial chromosome or some other replicating DNAelement. A second selectable marker that is not part of the FRT segment(Amp^(R)) provides a means to detect suboptimal plasmid preparation oranomalous recombination events.

The scheme for generating target strains with pEAW127 is described inFIG. 2, with details presented in Methods. FIG. 2 is a scheme forintroducing FRT sites into the bacterial chromosome. The plasmid pEAW127(or pEAW133) is processed to remove the replication origin. There-circularized DNA is then electroporated into prepared cells. Withoutthe replication origin, Kan^(R) is conferred on the cell only if atransposition event occurs transferring the FRT and Kan^(R) -containingsegment to the chromosome. The remainder of the electroporated DNAmolecule, including the transposase gene and the Amp^(R) marker, islost. The transposed segment is structured to facilitate the sequencingof the chromosomal DNA flanking the segment after transposition,allowing the target to be mapped to base pair precision within the E.coli genome database. The plasmid origin is first removed by cleavingwith EcoRI and re-ligating. Cleaving again with BspLU11I linearizes anyplasmids that retain the origin segment. The circularized plasmids areelectroporated into prepared E. coli cells. Transposition of the FRTsegment to the chromosome (or a replicating extrachromosomal element) isdetected by selection for Kan^(R). Anomalous recombination events ortransformation with intact pEAW127 can be eliminated by screening forAmp^(S). These generated a total of 6847 Kan^(R) colonies. Of 214Kan^(R) colonies screened (24 each from 9 different electroporations),only 9 (4%), were Amp^(R). The Amp^(R) colonies almost invariably arosefrom pEAW127 DNA from which the ori sequences had not been removed.

The FRT segment that is retained after transposition has short Tn5 ends(56 bp including the 19 bp required to direct transposition). The shortlength of these repeated end sequences facilitates the sequencing offlanking DNA in order to locate the transposed FRT site with base pairprecision. Sequencing primers can be directed at unique sequences in thetransposed segment, with sequencing directed outward across the outsideends. A number of the chromosomal segments containing FRT sites weresubcloned and sequenced, and the chromosomal positions of the FRT inseven independently chosen target strains are given in FIG. 3.

FIG. 3 diagrams the mapped locations of some FRT targets generated bypEAW127. The base pair locations of these targets are given in Methods.The bacterial strain used was MG1655 ΔrecATet^(S) /pLH29.

A variant of pEAW127 (designated pEAW133, FIG. 1) has been developedwhich includes a second FRT site on the opposite end of the Kan^(R)element from the first (FRT2 segment). Once the FRT2 segment istransposed to the chromosome, and the Kan^(R) element has been used tofacilitate selection and sequence-based mapping, the two FRT sitespermit the deletion of the Kan^(R) element while retaining thechromosomal FRT. This simply requires induction of FLP recombinase withIPTG, growth for a few generations without Kan, and screening for aKan^(S) colony. This feature should be useful in some applications inthat it preserves Kan^(R) selection for subsequent cloning steps. FLPhas previously been used in a similar strategy to remove selectablemarkers after gene disruption (5).

(iii) Targeting trials. The plasmid pEAW116 features a selectable marker(Tet^(R)) and a polylinker for cloning in a segment flanked by FRTsites. An Amp^(R) element in the remaining DNA again functions as amarker for anomalous events. Use of this plasmid (FIG. 4) is similar tothat outlined above for pEAW127. The plasmid can first be linearizedwith FspI. FIG. 4 is a scheme for targeting exogenous DNA to thechromosomal FRTs, using pEAW116 and derivatives. The plasmid pEAW116 (ora derivative like pEAW118) is processed to remove the replication originand Amp^(R) marker. The re-circularized DNA is then electroporated intoprepared cells. Without the replication origin, Tet^(R) is conferred onthe cell only if the electroporated circle is integrated into thechromosomal FRT in an FLP-mediated reaction. A targeted integrant shouldbe Amp^(s). Events resulting from improperly prepared DNA or anomalousrecombination are generally detected as Amp^(R). This step, not shown inFIG. 4, removes a fragment containing the origin of replication and partof the Amp^(R) element, and ultimately reduces the background of cellstransformed with unprocessed plasmid. Incubation of the larger linearfragment with FLP recombinase in vitro leads to product circlescontaining only one FRT along with the polylinker and Tet^(R). Thesecircles are then electroporated into the prepared target cells. Sincethe circles lack a replication origin, they are not retained unless theyare integrated into a replicating DNA molecule, and the potentialcomplexity of introducing a second replication origin into thechromosome is avoided. To illustrate the use of pEAW116, a variant wasconstructed (pEAW118) in which the recA+ gene and its regulatoryelements were cloned into the pEAW116 polylinker.

Virtually all of the Tet^(R) colonies arose from FLP-mediated targetedintegration. In trials with pEAW118, the production of Tet^(R) colonieswas reduced by at least two orders of magnitude if either thechromosomal FRT or the FLP-expressing plasmid was not present (Table 1).Of 265 Tet^(R) colonies screened over the course of four independenttargeting trials, 100% were also Amp^(S), indicating that the inclusionof unwanted plasmid sequences or transformation by unprocessed pEAW118did not constitute a significant problem. The cells used in these trialscontained a deletion of the recA gene. Interestingly, the targetingtrials showed that target #3 (FIG. 3), located in the nagC gene,exhibits a lower than normal targeting efficiency. This may define arelatively "cold spot" for FLP-mediated targeting in the E. coli genome.Even here, however, it was not difficult to obtain significant numbersof targeted integrants with pEAW118.

The site-specificity of targeting is illustrated in the Southern blotsin FIG. 5. FIGS. 5A and B is a demonstration of FLP-mediatedsite-specific chromosomal targeting with the FLIRT system. The bacterialstrain used was MG1655 ΔrecATet^(S) /pLH29. Panels A and B show Southernanalyses of successive steps in targeting. The probe in each case isdirected at genome sequences immediately adjacent to the FRT target.Genomic DNA was digested with PvuII in both cases. Referring to FIG. 5,the lanes are: "M," markers generated from a HindIII digest ofbacteriophage 1 DNA from New England Biolabs, (DNA fragment sizes areindicated in bp); "1," bacterial strain MG1655 ΔrecATet^(S) /pLH29without a target FRT; "2," after introduction of FRT target #3 (panel A)or 44 (panel B); "3," after targeting with pEAW118; "4," afterFLP-mediated excision of the pEAW118-derived DNA from the chromosome.Panel C shows the sensitivity to UV irradiation of the strains analyzedin panel B, lanes 1-4. Note the elevated resistance observed for thestrain from lane 3, reflecting the introduction of a wild-type recλgene. A probe was directed at genomic sequences adjacent to thechromosomal FRT. Upon introduction of the chromosomal FRT bytransposition, the labeled fragment is seen to increase in size by anincrement consistent with the introduction of the 1430 bp elementderived from pEAW127 (including the FRT plus Kan^(R) ; lane 2 in panelsA and B). The use of pEAW118 as the source of exogenous DNA adds another4270 bp when the chromosomal FRT is targeted (lane 3 in panels A and B).Targeting is efficient and reliably site-specific. Six colonies in whichprocessed pEAW118 was integrated into target #4 were selected at random,and examined by Southern analysis. The targeting occurred at the samelocation in each case (FIG. 5C). The targeted integration was alsoreversible (lane 4 in panels A and B). As shown in FIG. 5D, theintroduction of the DNA from pEAW118 introduces a degree of resistanceto UV irradiation that is consistent with the introduction of the recAphenotype into the cell. This phenotype is lost when the targeted DNA isexcised.

A potential problem with the use of pEAW116 is the need for processingwith the FLP recombinase, which is not yet commercially available¹. Wehave constructed a variant of pEAW116 in which all XbaI restrictionsites, other than those present in the FRT sites, have been removed. Theplasmid is pEAW135 (not shown, essentially identical to pEAW116), and itallows the removal of the replication origin by cleavage with XbaIfollowed by circularization (by ligation) of the fragment containing theTet^(S) element plus any cloned DNA. If the cloned DNA does not containan XbaI site, this alternative eliminates the need for FLP recombinase.

(iv) Integrant stability. To study the reversibility of thesite-specific integration event in more detail, integrants obtained witha precursor of the FLIRT system (20, 21) were examined to determine therate of integrant excision. Colonies resulting from a λFRT36 (21) Xchromosomal FRT integration reaction were isolated, and grown in L brothwith 1 mM IPTG. The FLP recombinase was expressed using pLH29, which isalso used in the FLIRT system. At intervals, cells recovered were platedon both LB plates and selective media. A typical result is shown in FIG.6. We also ran controls where no IPTG was added. The ratio of survivingintegrants/total cells decreased sharply in the first 6 generations ofgrowth in IPTG-containing media. Later, the decrease seemed to slowdown. The estimated excision rate during the first six generations wasabout 30% per generation on average, with a range of 25% to 40% pergeneration for 6 independent excision experiments. There was nodetectable excision when the integrants were grown in media without IPTG(FIG. 6). FIG. 6 graphs rates of FLP-mediated excision of targeted DNAfrom a chromosomal FRT site. Experiments were carried out as describedin Methods. The presence or absence of IPTG is indicated. The targets A,B, C, and CrecA- each have a single chromosomal FRT in CSH26, with thelast of these transduced to recA56. The FRT sites were introduced onbacteriophage l/Tn5 vectors using strategies described elsewhere (20,21). Targets A, B, and C correspond to targets 3601, 3602, and 3621,respectively, described previously (20, 21). FLP expression was providedby pLH29. All of the strains were originally targeted with λFRT36 (21),and it is the precise excision of this DNA element that is monitored inthis experiment.

To determine if integrants that survived after 25 generations wereresistant to excision, the cells that remained Amp^(R) were isolated andgrown in fresh media with 1 mM IPTG. The integrated DNA in these cellscan be excised as efficiently as the original integrants, again with anexcision rate about 30% per generation. We found no evidence for asubclass of cells in which excision was reduced or did not occur. Inaddition, cells in which the integrated DNA had been excised could besubjected to targeting trials again, and the apparent integrationfrequency of these cells was about the same as the apparent integrationfrequency of the parental target strains (data not shown). This resultshows that the targeting system based on FLP site-specific recombinationreactions is fully reversible and indicates that the FRT sites in thechromosome remain intact during repeated integration and excision. Wealso compared the excision rate of RecA+ and RecA⁻ integrants. The ratewas about the same in two independent trials, whether they had RecA+ orRecA⁻ phenotypes (FIG. 6).

Rates of excision were very similar in trials carried out withintegrants generated with the FLIRT system, using the protocol describedin Methods. However, we have recently found that most of the excisionoccurs in the stationary phase of cell growth rather than theexponential phase. If cultures are regularly diluted to maintainexponential growth, little excision is observed even in the presence ofIPTG. The simplest way to generate cells with the integrated DNAexcised, therefore, is to grow them up in an overnight culture and thenselect for excision immediately. In the absence of IPTG, little excisionis observed even in stationary phase cells.

(v) Effects of transcription from a nearby promoter. It is known thattranscription affects the topology of the DNA template, generatingpositive supercoils ahead of the RNA polymerase and negative supercoilsbehind (49). In addition, RNA polymerase might at least transientlyblock a chromosomal FRT site in its path during transcription. Todetermine how transcription might affect the efficiency of site-specifictargeting into the E. coli chromosome, we introduced our chromosomal FRTconstructs at a fixed position on the chromosome within the lac operon.We made two constructs. In the strain designated as MG1655lacZ-FRT, theFRT site is located within the lacZ gene, about 80 nucleotidesdownstream of the transcription initiation site. In the secondconstruct, designated as MG1655 lacY-FRT, the FRT site is located withinthe lacY gene. In this construct, the lacZ gene remains intact, and thetranscription from the lac promoter can be assessed by the expression ofthe lacZ gene product. The positions of FRT sites in these strains wereconfirmed by Southern analysis. To mediate targeting, both of thestrains contain the FLP expression plasmid pEAW38 (21). The expressionof the FLP recombinase on this plasmid is heat inducible. This allowedus to independently induce the lac-FRT operon by adding IPTG to themedium and FLP gene transcription by shifting the culture to hightemperature as needed.

Our results showed that transcription from the lac promoter did not havea dramatic effect on targeting frequency (Table 2). The apparentintegration frequency of MG1655lacZ-FRT slightly increased when 1 mMIPTG was added. When the FRT site was moved farther downstream to thelacY region, there was no detectable difference in integration frequencywith or without IPTG. These experiments have been repeated at least 8times for the lacZ-FRT construct and 3 times for lacY-FRT construct. Amodest effect of transcription (2-3 fold) was always observed forlacZ-FRT. We conclude that the FLP mediated integration is onlymoderately sensitive to transcription at the lac locus.

We have recently implemented new system refinements. We use a variant ofpEAW116 called pEAW135, in which all XbaI restriction sites (exceptthose in the FRT sites) have been removed. FIG. 7 diagrams pEAW135. Thisallows a user to remove the replication origin with XbaI enzyme. pEAW116involves the use of purified FLP recombinase and it would be anadvantage to use the widely available XbaI.

The construct shown in FIG. 7 does not eliminate the reliance of thesystem on the FLP recombinase. There are several steps needed to get DNAcloned into pEAW116 or its derivatives into the bacterial chromosome(referring to FIG. 4). In the first step, the plasmid must be processedin vitro by FLP recombinase to eliminate the left half of the plasmidsequences depicted in FIG. 1C (between the FRT sites at the top andbottom of the circle). The FLP recombinase reacts between the FRT sitesas indicated at the top of FIG. 4, creating a smaller circle containingthe sequences in the right half of the plasmid shown in FIG. 1C. Thiseliminates in particular the sequence called "ori", which would bedeleterious to the cell if introduced into the chromosome. The plasmidshown in FIG. 7 allows one to instead cut the plasmid at the two FRTsites (which both have a recognition site for XbaI), and then use DNAligase to make the same circle (including the right half of thesequences shown in FIG. 1C) that FLP recombinase is required for inpEAW116. This substitution of XbaI works only if there is no XbaI sitein the DNA a researcher may clone into the plasmid. In addition, oncethe processed DNA circles are introduced into the cell, introduction ofthe circle into the chromosome still depends on the presence of FLPrecombinase inside the cell (provided by expression from a plasmid suchas pLH29) in order to get the circle into the chromosome. (This stepshown about half-way down in FIG. 4.) This is true for both pEAW116 andfor the new FIG. 7 construct, pEAW135. The new construct simply getsaround the use of FLP recombinase in the test tube in at least somecases. In effect, there are two steps requiring FLP recombinase, oneoutside the cell (in vitro) and one inside the cell (in vivo). The newconstruct affects only the former (which is the only one requiring FLPrecombinase in a purified form).

3. Discussion

FLIRT is a plasmid-based system for FLP-mediated chromosomal targetingand genome rearrangement in E. coli. FRT sites can be introduced intothe chromosome of almost any E. coli strain, and mapped to base pairprecision. The chromosomal FRT sites are stable once introduced, sincethe Tn5 transposase gene is not included in the DNA transposed to thechromosome. The FRT can, in principle, be introduced at any chromosomallocation where Tn5 can transpose. Once on the chromosome, the FRTbecomes an integration site for exogenous DNA. The procedure forbringing in exogenous DNA makes use of a plasmid cloning vector,processed prior to electroporation to remove the replication origin.Chromosomal integration is efficient and reliably site-specific. Thetargeted integrants are stable as long as FLP recombinase is notinduced. However, integration is reversible in the presence of FLPrecombinase. Transcription initiation at an upstream promoter (Lac) hadonly a modest effect on targeted integration frequencies, although wecannot rule out the possibility that other promoters might affecttargeting to a greater extent.

A number of parameters that might affect targeting have been exploredpreviously, in some cases with FLP-based systems that are precursors ofFLIRT (20, 21). All of the recombination reactions are recA independent,and the FLP-mediated processes were several orders of magnitude moreefficient and reliable than events mediated by homologous geneticrecombination (20). There is no detectable pseudo-FRT site in the E.coli genome that could react with a normal FRT site, helping to ensurethat the background of anomalous recombination events is low. A surveyof 88 independently selected strains with chromosomal FRTs, placed onthe chromosome as randomly as can be done with Tn5 transposition,indicates that FLP-mediated chromosomal targeting is largely independentof the chromosomal location of the FRT site (20). However, we havedetected at least two apparent "cold spots" in the genome that alwaysgive 10 to 100-fold lower targeting frequencies than the others. First,4 of the 88 surveyed sites, all located within a 10,000 bp regionencompassing the cyo operon (20) gave lower than normal targetingfrequencies. The other 84 surveyed target sites were not mapped. Of the7 precisely mapped chromosomal FRTs used in the current study (FIG. 3),one located in the nagC gene (#3) also gave lower than normal targetingfrequencies. We do not know why these few chromosomal FRTs were lessefficient in targeting trials than normal, but targeting efficiency evenwith these was high enough that targeted integrants were easilyobtained.

A few features of the FLIRT system or applications of site-specificrecombination have been previously developed in other bacterial systems.First, a method for deleting a plasmid ColE1 origin in vivo by placingit between two phage f1 replication origins has been described (45). Theresult is a kind of suicide vector that can be used for chromosomalallele replacements. In the FLIRT system, provision has been made toremove plasmid replication origins enzymatically in vitro wherenecessary. The Cre-loxP system has been used to generate precisechromosomal deletions. Homologous recombination was used to positionloxP sites on either side of the DNA to be deleted, followed byinduction of the Cre recombinase on a suitable expression plasmid (1).Controlled deletion can be used to study gene function, and a similarapproach has been used in eukaryotic organisms in a range of studiesenumerated in the Introduction. The FLIRT system could expand the rangeof experiments that could be accomplished with such a precise deletionconstruct. If FRT sites were similarly positioned on either side of achromosomal bacterial gene or regulatory site [perhaps using the allelereplacement strategy of Slater and Maurer (45)], the FLIRT plasmid pLH29could be adapted to delete the DNA and pEAW116 or derivatives could beused to target variants of the same or different DNA segments to thesingle FRT that would be left behind at the same chromosomal location.Precise deletion with FLP has also been coupled to a conditionalreplication origin to permit the excision and amplification of largechromosomal segments in vivo, permitting their isolation as largeplasmids (33). The FLIRT system generally complements theseapplications.

Site-specific recombination may be usefully applied when the exogenousDNA has no homology to the bacterial genome, more precise control orhigher efficiency is required in the integration reaction to facilitatethe independent introduction of several alleles of a gene into anisogenic background, the exogenous DNA is required only transiently tofacilitate one step in strain construction, or a recA background isrequired for genetic complementation tests. Allelic or other geneticcomparisons can be made without the complication of chromosomal positioneffects. FLIRT simplifies the task of placing any DNA sequence directlyonto the chromosome. Parts or all of the system should be adaptable foruse in other bacterial species.

The technique should also facilitate the study of broader genomestructure. Sequences that readily take up altered DNA structures can bepositioned at a variety of locations in the chromosome and their effectson DNA or cellular metabolism studied. New replication origins,promoters, or termination sites for replication or transcription couldbe introduced. Although the use of Tn5 introduces the FRT sites intomore or less random locations in the chromosome, the FRTs can be placedmore precisely if a selection exists for the disruption of a particulargene. Note that the use of pEAW133 to introduce FRTs into the chromosomeallows for the simple removal of the Kan^(R) selection marker once theFRT site is mapped. This would set the stage for introducing a secondFRT site somewhere else in the same genome. Expression of FLPrecombinase would then lead to the inversion or deletion of theintervening genomic DNA. There are obviously many other possibilities.

4. Prophetic Applications of the FLIRT System

The FLIRT system makes use of natural transposition and site-specificrecombination systems to permit the reversible introduction of clonedDNA into defined sites on a bacterial chromosome. FLIRT will be veryuseful in basic and applied research involving bacteria. A series oflikely applications is summarized above.

Beyond these basic applications, we can imagine a variety of additionaluses in the future. A major effort is now underway to engineer bacteriaso that they possess new metabolic pathways useful to industry. Onepossibility is to create bacteria capable of efficiently degradingtoxins in chemical waste dumps or sewage treatment facilities.Introducing new metabolic processes to a bacteria would require theintroduction of the genes (DNA) encoding the enzymes required for theprocess. The introduction of new genes to a bacterial chromosome forthis purpose could be greatly simplified with FLIRT.

The FLIRT system is flexible and can be expanded to allow for quitecomplicated genetic constructs. The FRT site used by the FLP recombinase(at the heart of FLIRT) can be modified to produce multiple FRTs. Aminimal FRT site consists of two inverted 13 bp repeats, separated by an8 bp spacer. The FLP recombinase binds to the repeat sequences. The FRTcan be modified within the spacer region. The altered FRT sites would befunctional, but an FRT with a particular spacer sequence will react onlywith another FRT with exactly the same sequence. This property(described in Umlauf and Cox (1988) EMBO Journal 7:1945-1852) couldallow the introduction of several distinct FRT sites onto a bacterialchromosome. We could, for example, modify the FRT sites on the plasmidpEAW133. Once on the chromosome, each of these FRT sites could then beindependently targeted with cloned DNA. Hence, if one introducesdifferent modified FRTs at different places in the bacterial chromosome,one could target each independently by altering the FRT on the plasmidpEAW116 to be identical to the particular chromosomal FRT that onewished to target. This would allow the introduction of several distinctcloned DNA segments at distinct and definable locations about thechromosome.

Another application of FLIRT, or FLIRT components, would be in the areaof new antibiotics to treat bacterial infections. The recombinationsystems in FLIRT could be engineered into a delivery system to bring DNAencoding proteins toxic to the bacteria into their genomes. Such anapplication might use genetically-engineered bacteriophages, which couldprovide the mechanism needed to get the DNA into the bacteriaefficiently. The DNA itself would encode the toxic proteins and wouldalso include the elements of FLIRT needed to integrate the DNA into thebacterial genome. When the bacteria expressed the new DNA, it would, ineffect, kill itself.

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                  TABLE 1                                                         ______________________________________                                        The FLIRT system: Targeting efficiency with                                     pEAW118..sup.a                                                                        FLIRT System                                                                             No           No FLP                                        Experiment Complete chromosomal FRT recombinase                             ______________________________________                                        1         97         0            1.sup.b                                       2 63 0 1.sup.c                                                                3 23 0 0                                                                      4 82 0 0                                                                    ______________________________________                                         .sup.a The bacterial strain used was MG1655 ΔrecATet.sup.s /pLH29,      with FRT target #4 (FIG. 3). The four electroporation experiments were al     done on different days. Each used 0.03 mg of pEAW118 DNA, processed to        remove the replication origin and Amp.sup.R  marker as described in           Methods. For each experiment, separate sideby-side electroporation trials     were done with bacterial strains identical  #to MG1655                        ΔrecATet.sup.s /pLH29 except that they lacked either the chromosoma     FRT target or pLH29 (which expresses FLP recombinase). Numbers reflect th     total number of Tet.sup.R  colonies obtained in a trial.                      .sup.b Colony was Amp.sup.r.                                                  .sup.c Colony was Amp.sup.s.                                             

                  TABLE 2                                                         ______________________________________                                        The effect of transcription initiation from an                                  upstream promoter on targeting efficiency..sup.a                                        no. of colonies per plate.sup.b                                                              Ratio of +IPTB/-                                   Strain      +IPTG     -IPTG    IPTG (mean ± SD).sup.c                      ______________________________________                                        Non-target   1         0                                                        MG1655lacZ-FRT 139  39  242 ± 0.71 (n = 8)                                 MG1655lacY-FRT 206 247 1.11 ± 0.25 (n = 3)                               ______________________________________                                         .sup.a Introduction of exogenous DNA was accomplished by phage infection      rather than by electroporation. Results of a typical experiment are shown     using 2 × 10.sup.3  phage and about 108-109 cells per plate. Cells,     with an FRT site positioned into either the lacZ or lacY genes as noted,      were targeted with FRT36, a modified  phage with an FRT site and              selectable marker (21).                                                       .sup.b The average ratio of integration frequencies with or without           induction of transcription                                                    .sup.c n = number of independent experiments.                            

We claim:
 1. A method of introducing exogenous cloned DNA into abacterial chromosome in which the transposon Tn5 and the FLP recombinaseare functional in vivo, comprising the steps of:(a) introducing FLPrecombination target sites (FRTs) permanently at random locations in abacterial chromosome using a plasmid vector that contains an FRT withina modified transposon, two selectable markers, and a removablereplication origin; (b) mapping the introduced FRT; (c) cloningexogenous DNA into a plasmid vector comprising two FRT sites, twoselectable markers, and a removable replication origin; (d) removing thereplication origin in the vector of step (c); (e) introducing thealtered plasmid vector of step (d) into bacterial cells, wherein thebacteria cells comprise a functional FLP recombinase; and (f) obtainingtargeted integrants.
 2. The method of claim 1 wherein the transposon isa Tn5 transposon.
 3. The method of claim 1 wherein the bacteria is agram negative bacteria.
 4. The method of claim 2 wherein the bacteria isan E. coli.
 5. The method of claim 1 wherein the FRT is introduced intothe bacterial chromosome on a modified Tn5 transposon.
 6. The method ofclaim 1 wherein FLP recombinase is used in step (d) to remove the originof replication.
 7. The method of claim 1 wherein XbaI and DNA ligase areused in step (d) to remove the origin of replication.
 8. The method ofclaim 1 wherein more than one FRT is introduced into the bacterialchromosome.
 9. The method of claim 8 wherein the FRTs do not react witheach other.
 10. A plasmid comprising:a) at least one FRT site; b) anucleic acid sequence encoding a selectable marker located between twooutside ends of transposon Tn5; and c) a removable replication originand d) a gene for the Tn5 transposase located outside the sequencedefined in a) and b).
 11. A plasmid comprising:a) at least one FRT site;b) a nucleic acid sequence encoding a selectable marker located betweentwo outside ends of transposon Tn5; and c) a gene for the Tn5transposase located outside the sequence defined in a) and b), whereinthe plasmid also comprises:i) at least one other FRT site wherein theFRT sites define boundaries of two plasmid segments, segment a andsegment b; ii) nucleic acid sequences encoding two selectable markers,wherein one marker is in segment a and one marker is in segment b; iii)a removable replication origin in segment b; and iv) at least onerestriction site positioned for use as a cloning site in segment a whichleaves both selectable markers intact, wherein the restriction site ispart of a polylinker providing at least two restriction sites.